The alarming rise in serious antibiotic-resistant bacterial infections is generally acknowledged as a public health crisis. Of the estimated two million hospital infections in the United States in 2004, 70% were resistant to at least one antibiotic. Gram-positive bacteria belonging to three genera (staphylococcus, streptococcus and enterococcus) together cause more than 60% of all bloodstream infections (Wisplinghoff et al., 2004) and have acquired multi-drug resistance (e.g., methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE)), thereby causing major medical and, consequently, economic problems. This trend is largely attributed to the indiscriminate use of antibiotics in the medical and veterinary field, which has greatly accelerated the accumulation and exchange of genetic information coding for antibiotic resistance in pathogenic bacteria (Dancer, 2004).
Despite the urgent need for the development of new antibiotics, the major pharmaceutical companies appear to have lost interest in the antibiotic market. In 2002, only five out of the more than 500 drugs in phase II or phase III clinical development were new antibiotics. In the last six years, only ten antibiotics have been registered and only two of those did not exhibit cross-reactivity with existing drugs (Spellberg et al., 2004). This trend has been attributed to several factors, e.g., the cost of new drug development and the relatively small return on investment that infectious disease treatments yield compared to drugs against hypertension, arthritis and lifestyle drugs, e.g., for impotence. Another contributing factor is the increasing difficulty in finding new targets, further driving up development costs. Therefore, investigation into novel therapies or preventative measures for multi-drug-resistant bacterial infections is urgently needed to meet this impending healthcare crisis.
Active immunization with vaccines and passive immunization with immunoglobulins are promising alternatives to classical small molecule therapy. A few bacterial diseases that once caused widespread illness, disability and death can now be prevented through the use of vaccines. The vaccines are based on weakened (attenuated) or dead bacteria, components of the bacterial surface or on inactivated toxins. The immune response raised by a vaccine is mainly directed to immunogenic structures, a limited number of proteins or sugar structures on the bacteria that are actively processed by the immune system. Since these immunogenic structures are very specific to the organism, the vaccine needs to comprise the immunogenic components of all variants of the bacteria against which the vaccine should be protective. As a consequence thereof, vaccines are very complex, take long and are expensive to develop. Further complicating the design of vaccines is the phenomenon of “antigen replacement.” This occurs when new strains become prevalent that are serologically and, thus, antigenically distinct from those strains covered by the vaccines.
Direct administration of therapeutic immunoglobulins, also referred to as passive immunization, does not require an immune response from the patient and, therefore, gives immediate protection. In addition, passive immunization can be directed to bacterial structures that are not immunogenic and that are less specific to the organism. Passive immunization against pathogenic organisms has been based on immunoglobulins derived from sera of human or non-human donors. However, blood-derived products have potential health risks inherently associated with these products. In addition, the immunoglobulins can display batch-to-batch variation and may be of limited availability in case of sudden mass exposures. Recombinant-produced antibodies do not have these disadvantages and thus offer an opportunity to replace immunoglobulins derived from sera.
Over the last decade, a variety of recombinant techniques have been developed that have revolutionized the generation of antibodies and their engineering. Particularly, the development of antibody libraries and display technologies, such as phage display, or more recently developed display technologies, such as ribosome, yeast and bacterial display, have greatly influenced antibody preparation. In general, the established generation of antibody libraries in phages includes the cloning of repertoires of immunoglobulin genes or parts thereof for display on the surface of the phages. The starting material for preparing antibody libraries has been RNA isolated from the total population of peripheral blood lymphocytes or B cells from immunized or non-immunized donors. A problem associated with the use of the total population of peripheral blood lymphocytes or B cells for preparing antibody libraries is that functionally relevant and therapeutically effective antibodies against pathogenic organisms such as bacteria are underrepresented in these libraries.
This problem has now been solved by using RNA from a subset of antibody-producing B cells, i.e., IgM memory B cells, for the production of antibody libraries. Pathogenic organisms are known to have evolved many evasive techniques to avoid detection or attack from the immune system. For example, many bacteria display huge variation in their surface antigens or at least the antigenic sites on which the immune system focuses. Therefore, antibodies designed to protect against these bacteria should be capable of recognizing many antigens to provide the maximum coverage of the most common infections; however, because of extensive antigen variation, coverage of all strains of a type of bacterium by an antibody is difficult to accomplish. Furthermore, although antibodies that are cross-reactive between strains are required, antibodies that are additionally cross-reactive between species of bacteria are preferred as these would be more attractive to develop and use clinically.
T lymphocyte help is known to be an important feature of adaptive immunity. Activated by vaccination or infection, adaptive immune responses are directed against a limited set of immunogenic epitopes in a process that takes weeks to fully develop. Once complete, a population of memory B cells that have switched their surface immunoglobulin receptor from M to another subtype, e.g., G (switched memory B cells or alternatively called IgG memory B cells), is generated and primed to respond with the secretion of a variety of high-affinity protective antibodies specifically against the infectious organism responsible for the initial infection or for which the vaccination was carried out.
In contrast, innate immunity refers to defense mechanisms that a host mounts immediately or within several hours after exposure to antigen expressed by a pathogen (Germain, 2004). Unlike adaptive immunity, innate immunity does not have the capacity to recognize every possible antigen presented to it. Instead, it has evolved to recognize a few highly conserved structures present in many different microorganisms. Memory B cells expressing the immunoglobulin M surface receptor (IgM memory B cells) behave more like an arm of innate immunity. They are stimulated independent of T cell help and develop and mutate their immunoglobulin genes during early childhood (<2 years of age).
The end result of this process is a diverse and protective pre-immune repertoire that is capable of responding immediately to a wide variety of pathogenic organisms and is particularly important in protection against encapsulated bacteria. Thus, libraries constructed from immunoglobulin genes derived from IgM memory B cells comprise an antibody repertoire applicable to potentially all pathogenic organisms, regardless of the infection and vaccination history of the donors, and would give rise to a new generation of antibodies suitable for combating the growing problem of pathogenic organisms such as bacteria.
IgM memory B cell-derived immunoglobulin libraries have the added advantage that it is not necessary to have access to donors with specific infections, which in some cases, such as with emerging infectious diseases, may be difficult to locate and recruit. Moreover, making antibody libraries from RNA obtained from IgM memory B cells reduces the library size needed to encompass the entire functionally relevant repertoire. IgM memory B cells comprise only around 25% of the total B cell population and further contain less immunoglobulin mRNA than circulating blast cells and, thus, may be further underrepresented in a total B cell library. Moreover, the antibody libraries derived from RNA obtained from IgM memory B cells only comprise mutated heavy and light chain variable region sequences and do not comprise germline-encoded antibody products, meaning that the libraries are focused on the most functionally relevant antibodies that have gone through a maturation process.